High-quality insect genomes are essential resources to understand insect biology and to combat them as disease vectors and agricultural pests. It is desirable to sequence a single individual for a reference genome to avoid complications from multiple alleles during de novo assembly. However, the small body size of many insects poses a challenge for the use of long-read sequencing technologies which often have high DNA-input requirements. The previously described PacBio Low DNA Input Protocol starts with ~100 ng of DNA and allows for high-quality assemblies of single mosquitoes among others and represents a significant step in reducing such requirements. Here, we describe a new library protocol with a further 20-fold reduction in the DNA input quantity. Starting with just 5 ng of high molecular weight DNA, we describe the successful sequencing and de novo genome assembly of a single male sandfly (Phlebotomus papatasi, the main vector of the Old World cutaneous leishmaniasis), using HiFi data generated on the PacBio Sequel II System and assembled with FALCON. The assembly shows a high degree of completeness (>97% of BUSCO genes are complete), contiguity (contig N50 of 1 Mb), and sequence accuracy (>98% of BUSCO genes without frameshift errors). This workflow has general utility for small-bodied insects and other plant and animal species for both focused research studies or in conjunction with large-scale genome projects.
Organization: PacBio, University of Notre Dame, University of California, Davis